cytosine deaminase (CDase, EC 3.5.4.1) catalyzes the hydrolysis of cytosine to uracil by the following reaction. ##STR1## The enzyme, which plays an important role in microbial pyrimidine metabolism (O'Donovan and Neuhard, 1970), has been isolated from several different microorganisms, but does not appear to be present in mammalian cells (Nishiyama et al., 1985).
The physical properties of CDase from various organisms have been shown to differ significantly in terms of molecular weight, stability, and subunit composition. For example, CDase from Salmonella typhimurium has been purified to homogeneity (by SDS-PAGE) and is composed of 4 subunits of 54 kilodaltons (kDa) each (West et al., 1982) while the enzyme from Escherichia coli has a molecular weight of 200 kDa and is composed of 35 and 46 kDa subunits (Katsuragi et al., 1986). Both of these enzymes are highly thermostable, and maintain high activity at 55.degree. C.
Bakers' yeast (Saccharomyces cerevisiae) has also been used as a source for CDase. CDase previously obtained therefrom has a molecular weight of 34 kDa as determined by gel filtration (Ipata et al., 1971, 1978) and 32-33 kDa as determined by SDS-PAGE and amino acid analysis (Yergatian et al., 1977). The CDase enzyme that has been previously isolated from bakers' yeast therefore appears to be a monomeric protein.
Solutions of previously isolated bakers' yeast CDase maintain activity for at least 48 hr when stored at 4.degree. C. between pH 5-9 (Ipata et al., 1971, 1978). However, at 37.degree. C., a crude preparation of bakers' yeast CDase has been shown to lose half of its activity in 1 hr (Kream and Chargaff, 1952), and a purified form of the enzyme has a half-life of 30 min (Katsuragi, 1988). The half-life at 37.degree. C. can be increased to 28 days by immobilizing the enzyme onto epoxy-acrylic beads (Katsuragi et al., 1987). Thus, the thermal instability of CDase from bakers' yeast, along with its low molecular weight, distinguish it from the bacterial enzymes described earlier.
CDase has been used therapeutically for the conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU) (Katsuragi et al., 1987;Nishiyama et al., 1985; Sakai et al., 1985; Senter et al., 1987). However, bacterial sources of CDase are impractical for such use, requiring large-scale cultivation in order to obtain adequate activity (Sakai et al., 1985). Additionally, microbial extracts can cause undesirable side effects in recipients thereof.
Yeast can be used as a source of CDase to overcome these problems. However, the thermal instability of the previous yeast-derived product requires that the enzyme be immobilized prior to its use (Katsuragi et al., 1987). Thus, the isolation and purification of a thermally stable yeast CDase provides an improved enzyme for use in anticancer therapy. Similarly, cloning the gene for thermally stable CDase from yeast permits the introduction of defined alterations or additions to the gene itself, sequences controlling its expression, and gene fusions created between the gene and other molecules. Such novel constructs increase the efficiency or usefulness of the enzyme in anticancer therapy.